---
title: "5. Indel signature analysis"
author: "Lea Jopp-Saile and Daniel Huebschmann"
date: "28/01/2019"
vignette: >
  %\VignetteIndexEntry{5. Indel signature analysis}
  %\VignetteEngine{knitr::rmarkdown}
  %\VignetteEncoding{UTF-8}  
output: 
  BiocStyle::html_document:
    toc: yes

references:
- author:
  - family: Alexandrov
    given: LB
  - family: Nik-Zainal
    given: S
  - family: Wedge
    given: DC
  - family: Aparicio
    given: SA
  - family: Behjati
    given: S
  - family: Biankin
    given: AV
  - family: Bignell
    given: GR
  - family: Bolli
    given: N
  - family: Borg
    given: A
  - family: Borresen-Dale
    given: AL
  - family: Boyault
    given: S
  - family: Burkhardt
    given: B
  - family: Butler
    given: AP
  - family: Caldas
    given: C
  - family: Davies
    given: HR
  - family: Desmedt
    given: C
  - family: Eils
    given: R
  - family: Eyfjörd
    given: JE
  - family: Greaves
    given: M
  - family: Hosoda
    given: F
  - family: Hutter
    given: B
  - family: Ilicic
    given: T
  - family: Imbeaud
    given: S
  - family: Imielinski
    given: M
  - family: Jäger
    given: N
  - family: Jones
    given: DT
  - family: Jones
    given: D
  - family: Knappskog
    given: S
  - family: Kool
    given: M
  - family: Lakhani
    given: SR
  - family: Lopez-Otin
    given: C
  - family: Martin
    given: S
  - family: Munshi
    given: NC
  - family: Nakamura
    given: H
  - family: Northcott
    given: PA
  - family: Pajic
    given: M
  - family: Papaemmanuil
    given: E
  - family: Paradiso
    given: A
  - family: Pearson
    given: JV
  - family: Puente
    given: XS
  - family: Raine
    given: K
  - family: Ramakrishna
    given: M
  - family: Richardson
    given: AL
  - family: Richter
    given: J
  - family: Rosenstiel
    given: P
  - family: Schlesner
    given: M
  - family: Schumacher
    given: TN
  - family: Span
    given: PN
  - family: Teague
    given: JW
  - family: Tokoti
    given: Y
  - family: Tutt
    given: AN
  - family: Valdes-Mas
    given: R
  - family: van Buuren
    given: MM
  - family: van't Veer
    given: L
  - family: Vincent-Salomon
    given: A
  - family: Waddell
    given: N
  - family: Yates
    given: LR
  - family: Australian Pancreatic Cancer Initiative
    given: 
  - family: ICGC Breast Cancer Consortium
    given: 
  - family: ICGC MMML-Seq Consortium
    given: 
  - family: ICGC PedBrain
    given: 
  - family: Zucman-Rossi
    given: J
  - family: Futreal
    given: PA
  - family: McDermott
    given: U
  - family: Lichter
    given: P
  - family: Meyerson
    given: M
  - family: Grimmond
    given: SM
  - family: Siebert
    given: R
  - family: Campo
    given: E
  - family: Shibata
    given: T
  - family: Pfister
    given: SM
  - family: Campbell
    given: PJ
  - family: Stratton
    given: MR
  container-title: Nature
  id: Alex2013
  issued:
    month: 08
    volume: 500
    year: 2013
  publisher: Nature Publishing Group
  title: 'Signatures of Mutational Processes in Cancer'
- author:
  - family: Alexandrov
    given: LB
  - family: Kim
    given: J
  - family: Haradhvala
    given: NJ
  - family: Huang
    given: MN
  - family: Ng
    given: AW
  - family: Boot
    given: A
  - family: Covington
    given: KR
  - family: Gordenin
    given: DA
  - family: Bergstrom
    given: E
  - family: Lopez-Bigas
    given: N
  - family: Klimczak
    given: LJ
  - family: McPherson
    given: JR
  - family: Morganella
    given: S
  - family: Sabarinathan
    given: R
  - family: Wheeler
    given: DA
  - family: Mustonen
    given: V
  - family: Getz
    given: G
  - family: Rozen
    given: SG
  - family: Stratton
    given: MR
  container-title: Nature
  id: Alex2020
  issued:
    month: 02
    year: 2020
  publisher: Nature
  title: 'The Repertoire of Mutational Signatures in Human Cancer'
- author:
  - family: Alexandrov
    given: LB
  - family: Nik-Zainal
    given: S
  - family: Wedge
    given: DC
  - family: Campbell
    given: PJ
  - family: Stratton
    given: MR
  container-title: Cell Reports
  id: Alex_CellRep2013
  issued:
    year: 2013
  title: > 
    Deciphering signatures of mutational processes operative in human cancer
- author:
  - family: Sing
    given: Tobias
  - family: Sander
    given: Oliver
  - family: Beerenwinkel
    given: Niko
  - family: Lengauer
    given: Thomas
  container-title: Bioinfomatics
  id: ROCR_package2005
  issued:
      year: 2005
  title: >
    ROCR: visualizing classifier performance in R
---

```{r loadStyle, warning=FALSE, echo=FALSE, message=FALSE, results="hide"}
library(BiocStyle)
```

```{r packages, include=FALSE}
library(YAPSA)
library(Biostrings)
library(BSgenome.Hsapiens.UCSC.hg19)
library(knitr)
opts_chunk$set(echo=TRUE)
opts_chunk$set(fig.show='asis')
```

# Introduction: PCAWG signatures {#introduction}

In May 2018 a new and extended set of mutational signatures was published as a 
preprint by the Pan-Cancer Analysis of Whole Genomes (PCAWG) consortium 
[@Alex2020]. In the following, we refer to that set of signatures as the PCAWG 
signatures. The underlying data is the largest available cohort of sequenced 
(Whole Genome Sequencing (WGS) and Whole Exome Sequencing (WES)) cancer 
samples to date and represents a substantial increase in statistical power as 
compared to [@Alex2013] and [@Alex_CellRep2013]. On this data set, de novo 
mutational signature discovery analysis was performed with two different 
algorithms of the Non-negative Matrix Factorization (NMF) family of algorithms, 
*SigPorfiler* and *SignatureAnalyzer* (Bayesian NMF). In total 65 SNV mutational 
signatures (abbreciated SBS for single base substitution) were found, 49 of 
which were called validated, either because of good consensus between the two 
calling algorithms or because of presence in an orthogonal sequencing 
technology. In addition to the SNV or SBS mutational signatures, that analysis 
for the first time had sufficient statistical power to also extract 17 Indel 
(ID) signatures, based on a classification of Indels into 83 features, and 
Doublet Base Signatures (DBS) based on somatic di-nucleotide exchanges. In this 
vignette, we present an example analysis with the new PCAWG Indel signatures. 
The setup and line of arguments is analogous to the 
[first vignette](http://bioconductor.org/packages/3.11/bioc/vignettes/YAPSA/inst/doc/YAPSA.html).
However, YAPSA doesn't provide functions for DBS signatures as mutation counts 
are often not sufficient for a deconvolution analysis.

As for the [COSMIC SNV mutational signatures](http://bioconductor.org/packages/3.11/bioc/vignettes/YAPSA/inst/doc/YAPSA.html),
the patterns for both the SNV and Indel PCAWG mutational signatures are stored 
in the software package and can be loaded into the workspace as follows: 

```{r loadPCAWGsigs}
data(sigs_pcawg)
```

For a precise description of all the data loaded by the above command, please 
refer to [2. Signature-specific cutoffs](http://bioconductor.org/packages/3.11/bioc/vignettes/YAPSA/inst/doc/vignette_signature_specific_cutoffs.html)


# Classification of Indels

A prerequisite for an analysis of Indel mutational signatures is a consistent 
classification system of Indels. This is non-trivial and critically depends on 
the choice of features, which have to include whether a given Indel is actually 
an insertion or a deletion, the size of the Indel,as well as the motif context 
in order to assess whether micro-homologies are present. In the classification 
proposed by the PCAWG consortium there are 83 features in total, which form 
five major groups: 

1. Deletions of 1 bp C/(G) or T/(A) in a repetitive context. Taking into 
account the motif context of the deletion, the mutational event is further 
classified into categories of 1, 2, 3, 4, 5 or larger or equal to 6 identical
nucleotide(s).
1. Insertions of 1 bp C/(G) or T/(A) in a repetitive context. Taking into 
account the motif context of the deletion, the mutational event is further 
classified into categories of 0, 1, 2, 3, 4,  or larger or equal to 5
identical nucleotide(s).
1. Deletions of 2 bp, 3 bp, 4 bp or more or equal to 5 bp in a repetitive 
context. Each deletion is classified in a context of 1, 2, 3, 4, 5 or larger
or equal to 6 times the same motif.
1. Insertions of 2 bp, 3 bp, 4 bp or more or equal to 5 bps in a repetitive 
context.  Each deletion is classified in a context of 0, 1, 2, 3, 4 or larger 
or equal to 5 times the same motif.
1. Deletions of 2 bp, 3 bp, 4 bp or more or equal to 5 bp in a partially 
repetitive context with microhomology at the breakpoints. This partially 
repetitive context is defined by motif length of minus 1 bp, 2 bp, 3 bp, 4 bps 
or more or equal to 5 bp, located before and after the break-points of the 
deletion.

Indel classifications in the range of 1 - 5 bp are assigned to individual 
categories, while all variants larger than 5bp are considered as one category.
Of note, microhomology is defined by partial sequence similarity between the 
motif of the respective Indel and the immediate neighbouring sequence context. 
In the following, we illustrate the microhomology classification with an 
example. We take a deletion of the motif **ATGCGA**, being more than 5 bp in 
length with microhomology, i.e., partially repetitive context of 4 bp at the 
breakpoint junction with the motifs **ATGC** upstream and **GCGA** downstream 
of the deletion. The annotated feature category is then **MH\_5+\_4bp**. 

In YAPSA, the function `plotExchangeSpectra_indel()` can be used to plot the 
nucleotide exchange spectra of samples and/or signatures. The representation is 
analogous to the function `plotExchangeSpectra()` for SNV mutational signatures, 
while the colors and feature annotation is taken from [@Alex2020].

```{r captionSpectra, echo=FALSE}
cap <-": Nucleotide exchange spectra of the Indel signatures ID3, associated 
        with tobacco smoking, and ID6, related to deficiencies in homologous 
        recombination repair."
```

```{r INDELsigExample, include=TRUE, fig.width=15, fig.height=6, fig.cap= cap}
plotExchangeSpectra_indel(PCAWG_SP_ID_sigs_df[,c(3,6)])
```

# PCAWG Indel signatures

The publication [@Alex2020] reports signatures decomposed with an NMF
approach (*SigProfiler*) and a Bayesian NMF approach (*SignatureAnalyser*). It
is worth noting that YAPSA only has mutational signatures decomposed with
SigProfiler integrated so far, of which the validated signatures are also found
with *SignatureAnalyser*. Signatures only decomposed with SignatureAnalyzer are
not part of YAPSA. Signature ID15 was excluded as none of the samples had a
contribution to this signature. For seven of the Indel signatures, underlying 
mutational processes were asserted. Indel Signatures ID1 and ID2 can be found 
across all entities, while others such as ID13 is associated with UV light 
exposure and is found exclusively in melanoma (cf. @Alex2020).

```{r INDELsigInfo}
current_caption <- paste0("Information on Indel mutational signatures.")
if(!exists("repress_tables"))
  kable(PCAWG_SP_ID_sigInd_df, row.names=FALSE, caption=current_caption)
```

# Example data: Genome of the Netherlands

In order to demonstrate the functionalities of the Indel signature analysis 
with YAPSA, a publicly available data set was chosen, the Genome of the 
Netherlands
(<http://www.nlgenome.nl/?page_id=9>) [release5](https://molgenis26.target.rug.nl/downloads/gonl_public/variants/release5/).
The data set is not related to cancer and instead contains germline variation 
in the genomes of the Dutch population. Hence no biological interpretation or 
conclusion of this analysis with respect to mutational processes should be 
made, it is of sole technical and demonstrative purpose.


# Supervised Indel signature analysis

## Loading example data

We first load the example data, which is stored in the software package 
YAPSA. The file $\texttt{GenomeOfNl_raw.rda}$ has been filtered to only contain 
Indel variant calls.

```{r loadGoNL}
data(GenomeOfNl_raw)
GenomeOfNl_raw <- GenomeOfNl_raw[, c(1,2,4,5)]
```

Optional columns are being removed from the loaded data, which results in a 
vcf-like (analogous to the variant calling format) dataframe with 
`r dim(GenomeOfNl_raw)[1]` variants.
The binary file above was created with the following R code:

```{r loadGoNLraw}
load_data_new <- FALSE
if(load_data_new){
  data <- data.frame(matrix(ncol = 8, nrow = 0))
  for(index in seq_along(1:22)){
              print(index)
              temp <- tempfile()
              file_path <- paste0("https://molgenis26.target.rug.nl/
                                  downloads/gonl_public/variants/release5/
                                  gonl.chr",
                                  index, ".snps_indels.r5.vcf.gz")
              download.file(file_path, temp)
       
              data <- rbind(data, read.table(gzfile(temp, paste0("gonl.chr",
                                                    index,
                                                    ".snps_indels.r5.vcf")),
                                             header=FALSE, sep="\t", 
                                             stringsAsFactors = FALSE))
              data <- data[grep("INDEL", data$V8),]
              unlink(temp)
            
  }
  colnames(data) <- c("CHROM", "POS", "ID", "REF", "ALT", "QUAL", 
                      "FILTER","INFO")

  GenomeOfNl_raw <- data[, c(1,2,4,5)]
}
```

The obtained dataframe will later be used it in order to build a mutational 
catalog for supervised analysis of mutational signatures with PCAWG Indel 
signatures.

```{r showsTopOfDf, echo=FALSE}
kable(head(GenomeOfNl_raw), caption="Head of VCF file 
      containing the GoNL INDEL data")
```

## Data Preprocessing 

`GenomeOfNl_raw` does not contain any patient identifier information. 
For the purpose of demonstrating the use of YAPSA Indel signature analysis, 
such an information on patient identity is necessary. To do so, 15 synthetic 
patient identifiers (PIDs) were generated and 50 +/- 20 Indels per PID were 
randomly sampled from the data frame `GenomeOfNl_raw`.

```{r randomizeDataSet, warning= FALSE}
seed = 2
set.seed(seed)
number_of_indels <- sample(c(30:70), 15,  replace = TRUE)

index=0
seed=3
set.seed(seed)
vcf_like_indel_lists <- lapply(number_of_indels, function(size){
  df_per_PID <- GenomeOfNl_raw[sample(nrow(GenomeOfNl_raw), size, 
                                      replace = FALSE), ]
  index <<- index+1
  df_per_PID$PID <- rep(paste0("PID_", index), length(size))
  df_PIDs <- df_per_PID[order(df_per_PID$CHROM),]
  return(df_PIDs)
  })

vcf_like_indel_df <- do.call(rbind.data.frame, vcf_like_indel_lists)
kable(head(vcf_like_indel_df), caption="Head of the vcf_like_df
      containing the subsampled GoNL Indel data")
```

Using the artificial `vcf_like_df` created above as input, the function 
`create_indel_mutation_catalogue_from_df()` then builds a mutational
catalog $V^{INDEL}$ containing the absolute feature counts ($n$) for each
patient. This functions is equivalent to the function
`create_mutation_catalogue_from_df()` for SNV data. Besides the input of type 
`vcf_like_df`, the function `create_indel_mutation_catalogue_from_df()` also 
requires the Indel signatures (`PCAWG_SP_ID_sigs_df`) loaded previously into 
the workspace.
The function `create_create_indel_mutation_catalogue_from_df()` is a wrapper of 
several other functions:

1. `attribute_sequence_contex_indel()` the function annotates to the 
`vcf_like_df` a sequence context of 10 bp down- and 60 bp upstream of every 
Indel variant. Additionally, the information whether the variant is an 
insertion or deletion and the length of the Indel is annotated to every Indel 
mutation.
1. `attribution_of_indels()` attributes each variant to one of the 83 classes of
features 
1. `create_indel_mut_cat_from_df()` carries out the counting in order to obtain 
a mutational catalog with the dimensions $n \times m$.

Please note that this pre-processing step takes longer than all other functions 
in YAPSA and may be time limiting.

```{r createMutationalCatalog, warning=FALSE}
vcf_like_indel_trans_df <- translate_to_hg19(vcf_like_indel_df,"CHROM")
mutational_cataloge_indel_df <- create_indel_mutation_catalogue_from_df(
  in_dat = vcf_like_indel_trans_df,
  in_signature_df = PCAWG_SP_ID_sigs_df)

kable(head(mutational_cataloge_indel_df[,1:5]))
```

## LCD Analysis 

The core function of YAPSA is the function `LCD` and its derived functions, 
which perform supervised mutational signature analysis. Here we describe how 
this class of functions has been extended in order to make this supervised 
signature analysis available to Indel mutational signatures. As previously 
described for 
[SNV mutational signatures](http://bioconductor.org/packages/3.11/bioc/vignettes/YAPSA/inst/doc/YAPSA.html), 
the deconvolution is based on a non-negative least squares (NNLS) algorithm. In 
order to reduce false positive calls, 
[signature-specific cutoffs](http://bioconductor.org/packages/3.11/bioc/vignettes/YAPSA/inst/doc/vignette_signature_specific_cutoffs.html) 
were introduced in YAPSA. Signature-specific cutoffs
are applied after a first decomposition with NNLS, and only signatures with 
computed contributions higher than their signature-specific cutoff are used in 
a second decomposition which then yields the exposure values. Signature-specific 
cutoffs take the different detectability of signatures into account, reflected 
by a lower signature-specific cutoff for signatures with a distinct pattern and 
a higher signature-specific cutoff for signatures with broader patterns.

Of note, mutational processes have been asserted to fractions of both the sets 
of SNV and Indel mutational signatures. There are overlaps between the asserted 
mutational processes, as some of them can indeed leave imprints on both the SNV 
and Indel mutational landscapes. Combining the information extracted with an 
analysis of mutational signatures from the different types of mutation may thus 
increase discriminatory power.

As described 
[here](http://bioconductor.org/packages/3.11/bioc/vignettes/YAPSA/inst/doc/vignette_signature_specific_cutoffs.html), signature-specific cutoffs were trained in a modified 
ROC analysis with the package `r CRANpkg("ROCR")` [@ROCR_package2005] using the 
results of the unsupervised discovery analysis as training data. As for the SNV 
mutational signatures, signature-specific cutoffs are stored in dataframes 
within the software package and can be loaded into the workspace:

```{r loadCutoffs, warning=FALSE}
data(cutoffs_pcawg)
```

As described in 
[2. Signature-specific cutoffs](http://bioconductor.org/packages/3.11/bioc/vignettes/YAPSA/inst/doc/vignette_signature_specific_cutoffs.html), the modified ROC analyses are parametrized by 
defining cost terms for punishing false-negative ($cost_{FN}$) and 
false-positive ($cost_{FP}$) findings separately, but the shape and minima of 
the cost functions depend only on the ratio between the cost for a 
false-negative finding divided by the cost for a false positive finding, the 
$cost_{factor}$ ($cost_{factor} = cost_{FN}/cost_{FP}$). Therefore, there is one 
set of optimal signature-specific cutoffs per chosen value of $cost_{factor}$.

Optimal signature-specific cutoffs are stored in data frames within the software 
package and can be loaded into the workspace as shown above. In these data 
frames every row corresponds to a different value of $cost_{factor}$. For Indel
signatures the optimal cost factor was identified to be 3, thus the third line 
in `cutoffPCAWG_ID_WGS_Pid_df` can be chosen for analysis.

```{r LCDdecompostion, warning=FALSE}
current_catalogue_df <- mutational_cataloge_indel_df 
current_sig_df <- PCAWG_SP_ID_sigs_df
current_cutoff_pid_vector <- cutoffPCAWG_ID_WGS_Pid_df[3,]
current_sigInd_df <- PCAWG_SP_ID_sigInd_df

current_LCDlistsList <- LCD_complex_cutoff_combined(
  current_catalogue_df,
  current_sig_df,
  in_cutoff_vector = current_cutoff_pid_vector,
  in_filename = NULL,
  in_method = "abs",
  in_sig_ind_df = current_sigInd_df)

current_consensus_LCDlist <- current_LCDlistsList$consensus
if(!exists("repress_tables")) 
  as.character(current_consensus_LCDlist$out_sig_ind_df$sig)
```

## Visualization

The exposures, i.e., the contributions of the different signatures to the 
mutational catalog of every sample, can be visualized with the function
`exposures_barplot()`. All the functionalities which are available for
SNV mutational signatures, such as the annotation of subgroups (cf. 
[1. Usage of YAPSA](http://bioconductor.org/packages/3.11/bioc/vignettes/YAPSA/inst/doc/vignette_confidenceIntervals.html)) 
are also available for the Indel mutational signatures.

```{r captionExposure, echo=FALSE}
cap <- ":Exposures to Indel mutational signatures in the artificial data created 
        by sampling GoNL variants. Exposures were obtained from
        a decomposition with PCAWG Indel signatures as well as their signature
        specific-cutoffs (cutoffPCAWG_ID_WGS_Pid_df)."
```

```{r plotExposure, echo=TRUE, warning=FALSE, fig.width=15, fig.height=6, fig.cap= cap}
exposures_barplot(current_LCDlistsList$perPID$exposures,
                  current_LCDlistsList$perPID$out_sig_ind_df)
```

# Confidence Intervals

In order to assess trustworthiness of the computed exposures, YAPSA provides 
the calculation of confidence intervals (CIs). Analogously to 
[CIs for SNV mutational signatures](http://bioconductor.org/packages/3.11/bioc/vignettes/YAPSA/inst/doc/YAPSA.html), 
the CIs for Indel mutational signatures are computed using the concept of 
profile likelihood.

```{r captionCI, echo=FALSE}
cap <- "Confidence interval calculation for exposures to Indel mutational 
        signatures"
```

```{r CI, echo=TRUE, warning=FALSE, fig.width=17, fig.height=15, fig.cap=cap}
confidence_intervals_ID <- confidence_indel_only_calulation(
  in_current_indel_df = current_catalogue_df)
plot(confidence_intervals_ID$p_complete_PCAWG_ID)
```

Note: With `confidence_indel_only_calulation()` only for INDELs the confidence
intervals are calculated. To calculate the confidence intervals for SNVs and
INDELs please use the function `confidence_indel_calulation()` and supply the 
mutational catalogs of both SNV and INDEL counts.

# References 
